pCAR and its uses

ABSTRACT

The invention provides improvements in the field of animal models for testing effects of genes introduced into animal cells or tissue by adenoviral gene transfer. More particularly the invention provides a plasmid construct that expresses a porcine adenovirus receptor (pCAR) and transgenic animals that show expression of pCAR.

[0001] The invention provides improvements in the field of animal models for testing effects of genes introduced into animal cells or tissue by adenoviral gene transfer.

[0002] Adenoviruses infect cells using two cell surface receptors, the “Coxsackie B and adenovirus 2 and 5 receptor” (hereinafter referred to as CAR; Bergelson J. M., et al, Science 275, 1320-23, 1997) and the integrin receptors (ανβ133 or ανβ5; Wickham, T. J. et al, Cell 73, 309-19, 1993) the contents thereof being incorporated herein by reference. Adenoviral based vectors are widely used in gene therapy, as they represent one of the most efficient ways to deliver genes to target cells. They are of particular interest for in vivo gene therapy proof-of concept experiments in rodent models. However, rodent tissues are not well transducible with adenoviral vectors.

[0003] In its broad aspect the invention is concerned with genetic modification of target cells which are normally refractory to adenoviral transduction. More particularly the invention provides a plasmid construct that expresses a porcine adenovirus receptor (PCAR) and transgenic animals that show expression of pCAR.

[0004] Organ transplants of liver, kidney, lung and heart are now regularly performed as treatment for endstage organ disease. Despite the use of modern immunosuppressive drugs acute and chronic graft (tissue or organ) rejection still remain major factors in graft loss. There is, therefore, a continued need for means to inhibit acute and chronic graft rejection and increase graft acceptance, e.g. through induction of peripheral tolerance without causing serious toxic side effects typically associated with conventional immunosuppressant therapy. When considering cell transplantation, e.g. bone marrow derived cells, islet cells, neuronal cells etc. one is faced with similar problems of rejection. Making organs or cells less immunogenic through genetic modification is seen as an alternative or add on to conventional immunosuppression.

[0005] Rodent animal models are of crucial importance for testing the immunomodulatory effects of new gene products. However in the case of using adenovirus as gene delivery vehicle rodent models have so far proven to be of limited value, as many rodent organs or cell types are refractory to adenoviral transduction. This may be due to the fact that either the adenoviral receptor CAR is not expressed or only weakly expressed on the cell surface of the cells of interest.

[0006] Accordingly, the invention provides a plasmid or vector construct that comprises a DNA molecule which expresses porcine CAR (SEQ ID NO:4ereinafter referred to as pCAR) or a biologically active fragment or derivative thereof, for example a C-terminally truncated porcine CAR (SEQ ID NO:2hereinafter referred to as ΔpCAR), that retains full functionality as adenoviral receptor.

[0007] pCAR comprises an intracellular domain, a transmembrane domain and a an extracellular domain that binds to the adenoviral fibre proteins, i.e. a total sequence of 365 amino-acids. It will be understood that any nucleic acid sequence encoding a porcine CAR homologue is a candidate for utilization in the present invention. For example, it may include a pCAR sequence with a modified, mutated or truncated region thereof, that retains the activity of mediating adenoviral transduction. It will be further understood by the skilled person that any nucleic acid sequence which encodes a biologically active form of pCAR, including but not limited to a genomic or cDNA sequence or functionally equivalent variant or mutant thereof or a fragment thereof which encodes a biologically active protein fragment or derivative which mediates adenoviral transduction, may be utilized in the present invention. For example, ΔpCAR may comprise the leader sequence of 19 amino-acids, the extracellular domain of 216 amino-acids, the transmembrane domain of 24 amino-acids and a truncated cytoplasmic domain, e.g. limited to 3 amino-acids. Two potential sites for N-glycosylation are located at Asn 106 and Asn 201. Amino-acids present in the sequence which are not essential to the activity may be changed by mutation, e.g. amino-acid 258 may be changed from Val to lie; amino-acid 262 may be changed from His to Arg.

[0008] Preferred nucleic acid sequence for use in the invention is e.g. as disclosed in SEQ ID NO: 1 from nucleotide 3229 to nucleotide 4014. The corresponding amino acid sequence encoded by such DNA sequence is indicated in SEQ ID NO:2.

[0009] Any known expression vector or plasmid that is capable of expression upon transfection of a specified eukaryotic target cell may be utilized to pratice the invention. “Plasmid” and “vector” can be used interchangeably in the present specification as the plasmid is the most commonly used form of vector. An expression vector is a vector capable of directing the expression of genes to which they are operatively linked. An operable linkage as used herein refers to the position, orientation and linkage between a structural gene and expression control element(s) such that the structural gene can be expressed in any host cell. The term “expression control element” includes promoters, enhancers, ribosome binding sites etc. Any eukaryotic promoter and/or enhancer sequences available to the skilled person which are known to control expression of the nucleic acid of interest may be used in plasmid vector constructs, including but not limited to a cytomegalovirus (CMV) promoter, a Rous Sarcoma (RVS) promoter, a Murine Leukemia (MLV) promoter, a herpes simplex virus (HVS) promoter, such as HSV-tk, a β-actin promoter, e.g. chicken β-actin, as well as any additional tissue specific or signal specific regulatory sequence that induces expression in the target cell or tissue of interest. A preferred expression vector or plasmid according to the invention is e.g. an eukaryotic expression vectors, e.g. a pβ-actin-p16PL vector such as p(chicken)β-actin-p16PL.

[0010] In one such embodiment, a DNA sequence encoding pCAR is subcloned into the DNA plasmid expression vector, e.g. pβ-actin-p16PL, resulting in pβ-actin-pCAR-p16PL. p16PL is a standard mammalian expression vector, containing a gene that encodes a selectable marker, e.g. an antibiotic resistance gene, and a β-actin promoter active in mammalian cells (K. M. Marsden et al, J. Neurosc., May 15, 1996,16(10): 3265-3273). Such a construct, which may be constructed by one of ordinary skill with components available from numerous sources, will drive expression of a pCAR DNA fragment ligated downstream of the β-actin promoter subsequent to transfection of the target cell. More specifically, pCAR is cloned from pig liver RNA using a PCR based approach. The PCR fragment is inserted into the expression vector pSport (Life Technologies). This plasmid serves as template to create the truncated version of ΔpCAR. Preferably pβ-actin is pβ-(chicken) actin.

[0011] The invention further provides host cells into which a recombinant expression vector of the invention has been introduced. A host cell can be any prokaryotic or eukaryotic cell, e.g. bacterial such as E. Coli, yeast or mammalian cells, e.g. CHO or COS cells.

[0012] The host cells of the invention may preferably be used to produce nonhuman transgenic animals, preferably a mammal, more preferably a rodent such as a rat or mouse, or a pig.

[0013] For example, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a pCAR-coding sequence has been introduced. A transgenic animal of the invention, more preferably a mammal, most preferably a rodent or a pig, may be created by introducing a pCAR expression construct into the male pronuclei of a fertilized oocyte, e.g. by microinjection, or into embryonic stem cells, e.g. by electroporation. Methods for generating transgenic rodents have become conventional in the art and are described e.g. in U.S. Pat. Nos. 4,736,866, 4,870,009, 4,873,191, or in Manipulating the Mouse Embryo, B. Hogan, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). For example the expression construct may be introduced into an embryonic stem cell line and cells in which the introduced pCAR gene has integrated are selected. The selected cells are then used to produce chimaeras with known standard procedures. A chimaeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. The pCAR expression plasmid may also be inserted into somatic/body cells of the donor animal to provide a somatic recombinant animal, from whom the DNA construct is not capable of being passed on to offspring. For example, a somatic cell from the transgenic animal can be isolated and induced to exit the growth cycle and enter G_(o) phase. The quiescent cell can then be fused, e.g. through the use of electrical pulses, to an enucluated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring of this female foster animal will be a clone of the animal from which the somatic cell is isolated. Or for example, an improved method of cloning pigs using donor nuclei from non-quiescent differentiated cells in which the desired DNA, e.g. porcine CAR or fragments or variants thereof, is inserted in said differentiated pig cell or pig cell nucleus. This improved method is described in U.S. Pat. No. 6,235,969 B1 and is hereby incorporated by reference.

[0014] The present invention also provides a method for improving adenoviral gene transfer in a rodent using a transgenic rodent which expresses or overexpresses pCAR. Such rodents may be used as models in gene therapy to test adenoviral transduction, e.g. prevention or treatment of acute or chronic graft rejection, autoimmune disorders, e.g rheumatoid arthritis, cardiovascular disorders, e.g. restenosis, nervous system disorders, e.g. parkinson disease, etc. A preferred embodiment of the invention is the use of such rodents expressing or overexpressing pCAR in transplantation experiments, for example, of organs, tissues or cells, e.g. lung, heart, kidney, liver, pancreas, small bowel, spleen, pancreatic islets, neuronal or stem cells, etc. For example, organs, tissues or cells of such transgenic rodents, e.g. mice, are removed, in vitro transduced with the adenoviral gene delivery vector to be tested and then transplanted into rodents, e.g. mice, e.g. such animals which do not express pCAR.

[0015] The functional expression of pCAR, e.g. ΔpCAR may also be used to generate transgenic pigs that overexpress this adenoviral receptor. Porcine organs, tissues or cells transgenically modified to express high levels of pCAR may be used as recipients for adenoviral gene therapy vectors. Such transgenic modified organs, tissues or cells can be transfected with adenoviral gene therapy vectors carrying thrapeutically beneficial genes either ex vivo or in vivo and can be subsequently transplanted in a recipient. Beneficial genes are those that are expected to confer graft protection following transplantation of these gene delivered organs in xenotransplantation therapy. The present invention comprises a method to generate such transgenic pigs expressing high levels of pCAR or a functionally equivalent variant or mutant thereof or a fragment thereof, e.g. as disclosed above, and gene therapy methods for preventing or inhibiting graft rejection in a recipient using organs, tissues or cells of such transgenic pigs.

[0016] Definitions

[0017] To facilitate understanding of the invention, a number of terms are defined below.

[0018] “Fragment” of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide. “Fragment” of a polynucleotide sequence refers to a polynucloetide sequence that is shorter than the reference sequence of SEQ ID NO: 2 and 4.

[0019] “Transduction” Transfer of genetic material or characteristics from one bacterial cell to another by the incorporation of bacterial DNA into a bacteriophage.

[0020] “Variant” refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains the essential properties thereof. A typical variant of a polynucleotide differs in nucleotide sequence from the reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from the reference polypeptide. Generally, alterations are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, insertions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. Typical conservative substitutions include Gly, Ala; Val, lie, Leu; Asp, Glu; Asn, GIn-I Ser, Thr; Lys, Arg; and Phe and Tyr. A variant of a polynucleotide or polypeptide may be naturally occurring such as an allele, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis. Also included as variants are polypeptides having one or more post-translational modifications, for instance glycosylation, phosphorylation, methylation, ADP ribosylation and the like. Embodiments include methylation of the N-terminal amino acid, phosphorylations of serines and threonines and modification of C-terminal glycines.

[0021] The following Exampes are illustrative only and not limiting of the invention. The β-actin promotor used in the Examples is the β-(chicken)actin promotor.

EXAMPLE 1 Construction of the Expression Vector

[0022] The full length cDNA for porcine CAR is cloned from pig liver using degenerated primers (forward: 5′-accatggcgckcctrctgt-3′ and reverse: 5′-catatggaggctytatacya-3′ in which k=g or t; r=a or g and y=c or t). The PCR fragment is bluntend inserted into the vector pSport (Life Technologies). Porcine CAR (SEQ ID NO:4) has an overall aminoacid homology of 91% to human as well as mouse CAR. This clone is used as template to generate the ΔpCAR gene as disclosed in SEQ ID NO:1 from nucleotide 3229 to nucleotide 4014, using PCR. The primers used to generate this construct contain two amino acid changes at the C-terminal end of the construct. The sense primer SpeI-CAR (5′-ggactagtgccaccatggcgctcctgctgtgcttc-3′) is located at position 1-21 of pCAR and contains a SpeI site, a Kozak sequence and the start codon. The antisense primer CAR-XbaI (5′-ctctagattaacgacagcaaaagatgataagacc3′) is located at position 760-786 of porcine CAR containing a stop codon and a XbaI site. The PCR amplification used the following conditions: 1× native Pfu buffer, 2.5 mM MgCl₂, 0.2 mM dNTPs, 2.5 U native Pfu polymerase (Stratagene) and 20 pmol SpeI-CAR and CAR-XbaI (each). Porcine CAR cDNA (5 ng) is used as template and hot start PCR is performed using the following profile: 1× (5 min 95° C.) 20× (30 sec 95° C., 1 min 55° C., 1 min 30 sec 72° C.) 1× (3 min 72° C.). A PCR product of a predicted size of 788 bps is obtained and separated on a 1% low melting agarose gel (SeaPlaque GTG; FMC). The band is excised and the PCR product isolated from the gel piece using the QlAquick gel extraction kit from Qiagen according to the manufacturers protocol. The isolated PCR product is then digest with XbaI (LifeTechnologies) and repurified as described above. The digested purified PCR product is ligated into MscI-XbaI digested pβactin-16PL vector.

[0023] INVaF′chemically ultracompetent bacteria from Invitrogen are transformed and 48 colonies picked, rescreened by PCR using SpeI-CAR and CAR-XbaI as primers. From 48 colonies analyzed 20 contain the insert—12 are selected for DNA sequencing. The sequencing primer actinsense (5′-accggcggggtttatatcttc-3′) is the 5′-primer located just upstream of the MCS of the pβactin-16PL vector. Actinanti (5′-cctctacagatgtgatatggc-3′) is the 3′-primer located just downstream of the MCS of pβactin-16PL vector. The nucleotide sequence of the β-actin promotor, the ΔpCAR gene and the SV40 polyadenylation signal is shown in SEQ ID NO:1.

EXAMPLE 2 In vitro Expression of ΔpCAR in Mammalian Cells (Westem Blot)

[0024] A human lung carcinoma cell (A30), rat embryonic fibroblasts (Rat2, ATCC:CRL-1764) and chinese hamster ovary cells (CHO) are used for transient transfections. Culture conditions are as follows: Cell Line Medium Serum Supplement Antibiotics A30 RPMI 10% FBS 1% NEAA 1% PS Rat2 DMEM 10% FBS 1% PS CHO αMEM 10% FBS 1% PS

[0025] In addition, all media contain 2 mM Glutamax II. Cultures are maintained at 37° C. in a water saturated air atmosphere containing 5%CO₂.

[0026] Cells are transfected with either the control plasmid (pβactin-16PL vector) or pβactin-ΔpCAR-16PL. In brief, an 80% confluent (approx. 1×10⁸ cells) 15 cm dish is transfected with 15 μg plasmid DNA using SuperFect from Qiagen according to the manufacturers protocol. After 24 h, cells are harvested, washed and cell pellet resuspended in 0.5 ml Lammli's buffer. Western blotting supplies are obtained from BioRad unless otherwise stated. Samples are sonicated for 10 sec, heat-denatured for 5 min at 95° C. and cellular debris removed by centrifugation (10 min 13 krpm Eppendorf). Samples are stored at −20° C. until further use. A quantity of 30 μl/lane is loaded on to a 12% denaturing polyacrylamide gel (SDS-PAGE) and run at 100V for 90 min in 1× Tris/Glycine/SDS buffer. Gel is then electrotransfered onto a 0.45 μm Protan BA85 (Schleicher&Schuell) nitrocellulose membrane in 1× Tris/Glycine buffer (Novex) containing 20% methanol. The membrane is blocked for 1 h in phosphate-buffered saline (PBS) containing 5% non-fat dry milk and 1%Tween 20 (Sigma), followed by 1 h incubation with an affinity-purified polyclonal chicken-anti human CAR antibody at 1:500 in blocking solution. In between antibody incubation steps the membrane is washed by two short rinses in PBS/1%Tween 20 followed by 2×15 min in the same washing buffer. The membrane is incubated for 1 h with a biotinylated rabbit-anti chicken IgY (Vector Laboratories) diluted at 1:1000 in blocking solution, followed by 30 min incubation with streptavidin-horseradish peroxidase (Vector Laboratories) at 1:1000 in blocking solution. Membrane is incubated for 5 min in enhanced chemiluminescence (ECL) substrate (Amersham), solution is carefully drained and membrane put in a Photogene Development folder (Life Technologies). ECL signals are detected by exposing Hyperfilm ECL (Amersham) to the membrane and films are developed on a X-Ray film developer (Agfa).

[0027] All 3 different cell lines which are transfected with ΔpCAR-16PL show an additional strong protein band which has the predicted molecular size. As a positive control 100 ng of recombinant human soluble CAR (hCAR) purified from E.coli source is used.

[0028] The polyclonal chicken-anti human CAR antibody used above are prepared as follows: A soluble version of human CAR is generated by PCR using the CAR1 (5′-accggccatggcatatggatttcgccagaa-3′ and the CAR2 (5′-accggctcgagagctttatttgaaggagggac-3′) primers. As template full length human CAR cloned from HeLa cells is used. The soluble human CAR PCR fragment is digested with NdeI and XhoI and inserted into the prokaryotic expression vector pET-17H, which contains a C-terminal histidine tag. The construct is transformed into bacteria and cells are induced to produce the soluble human CAR protein. The protein is purified by commonly used methods and is injected into an adult female chick. The eggs of the hen are collected and antibodies isolated from the egg yolk.

EXAMPLE 3 Functionality of ΔpCAR in Mammalian Cells (Adenoviral Gene Transfer)

[0029] The functionality is tested by transient transfection of CHO cells with the construct to be tested or the control plasmid, followed by transduction with an adenovirus which contains a reporter gene.

[0030] CHO cells are seeded into 24 well plate at a density of 12,000 cells/well. Cells are transiently transfected with 0.5 μg plasmid DNA of either pβactin-16PL or pβactin-ΔpCAR-16PL and incubated for 24 h. Cells are then transduced with an adenoviral vector carrying β-galactosidase as a reporter gene (moi 0-100) for 2 h. Virus solution is removed and cells incubated for an additional 4 days. Reporter gene expression is monitored using staining for nuclear β-galactosidase. Only ΔpCAR transfected cells are transduced with the reporter gene.

EXAMPLE 4 Generation of Transgenic Mice

[0031] (a) Generation of ΔpCAR BALB/c ES Cell Lines

[0032] 5×10⁶ BALB/c ES cells (“Efficient targeting of the IL-4 gene in a BALB/c embryonic stem cell line”, Noben-Trauth et al, Transgenic-Res. 1996 Nov; 5(6): 487-91) are electroporated with 30 μg of the linearized construct. Transfected cells are selected with G418 (200 μg/ml). G418-resistant clones are screened for integration events by PCR. The ES cells are lysed 1 h/37° C. with 20 μl Lysis buffer (PCR buffer 1×; SDS 1.7 μM; Proteinase K 50 μg/ml) heat inactivated 85° C./15 Min. and cleared by centrifugation. 1,3 μl lysed solution is used in for a 50 μl PCR. Positive clones are further verified by Southern analysis.

[0033] (b) Generation of ΔpCAR Transgenic Mice

[0034] BALB/c-ES cell clones carrying one ΔpCAR allele are injected into C57BU6 host blastocysts and transferred into pseudopregnant foster mothers according to standard protocols. Chimaeras are mated with BALB/c females and albino offspring (indicative for germ line transmission) are analyzed by PCR for target integration and Southern analysis. Heterozygous animals are generated by back-crossing of Fl animals to Balb/c wild type animals and Southern analysis of the F2 animals. The homozygous lines are established by mating heterozygous F1 animals.

EXAMPLE 5 Transplantation

[0035] Hearts of transgenic mice obtained according to Example 4 are removed, in vitro transduced by infusion with an adenovirus carrying β-galactosidase and then heterotopically transplanted into female mice (which do not express PCAR). Age matched Balb/c male mice are used as controls. 4 days after transplantation hearts are removed, perfusion stained for nuclear β-galactosidase, paraffin embedded and sectioned. Sectiones are counterstained with hematoxylin and evaluated by light microscopy. Positive expression for β-galactosidase is seen in the transgenic mice compared to the control animals.

1 4 1 4286 DNA porcine CDS (3229)..(4014) 1 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat 60 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgcca 120 agttgggatc tttgcattgg cccacggctc tcaggatggg gatgctcccc ttcagcaccc 180 ggttcccctt ggaaactgat ggtcctggct ctgtggcatg gcagtggcac tgtgaggagc 240 ccctaccagc agcacacagt gggtttggca ctgccacgct ccggatgccg cgctctgatc 300 caaccccata atcaagggaa cccgaattgc cccatcattg cccccaccac ccccatcctg 360 ccgggccctc acaccccacg ctgccttgtg gtgacattcc ccagcccaaa cccacggctt 420 catggctacc gcggggcatt tcccattgcc gccccattat cagctctgca cacctcccgc 480 tgtacccatg cctcgtggct gcccttcttt gacgtataat cttctaatta atacccggcc 540 ttgtcaaagt ggagcacaaa cgttaattaa ttccccagca ggcaggtaat taacagtgtg 600 actccctttt tgctgcgagt ggggctgata cagagagatg tggcactatg gagcccacgg 660 ggtcctggca ctgggtgccc acggaggtcc ccatgtgctg cagtgtcacc gcctccgagg 720 tgacagtatt gtccctgcgg tgtccctgca gctcagctct gtccacaggg ccacctccag 780 tttggagggg acacaatgca gccccgatgc aacccatcct cgcagcatcc cagggacaaa 840 gaccccactg caagaccgca cacagggctg ggtcccgctc ccctaatatc tacagtgctt 900 ttgcatggcc ccttaatcaa tgcagttaat cagcatgcgc tcatgcaccg ctctggagct 960 gcaaagcccc tcgcagcgct gctcaccaac accgcgcacc gccccggccc agcctgcagc 1020 acgcgctgca aacaggaaag aaacaaaata ttgcccaaat gtaggcaaag gcattcggct 1080 gccttgacct ccgccgggcc gggccctgcc tgactcagct ccttactcag cgctcgcttc 1140 ctccctccgg ctgccaccgc cgcagcgcac accctgacaa agagtggccc ttaacgggct 1200 ctgaggtgca cccagcagtg cactcagcag tccaagggcc ggcctggagg tttgcaccgc 1260 tacgtgctga cattagcatt gaacttggcc ctgggtagtg ctgcaggccg ggcggggtgg 1320 gtgtagagag tgcagcgcgc gttgcacccg gtgccccttc ccctcccttg catcccagca 1380 ggctgcaccc cagcaccagg cccgtgcatg catgctcctg gtgttattgc agcctggtgc 1440 atgcatgcgt cttagtggtg cagcgctgtg catgcatcct ccttggtgtg tagcagctta 1500 gtgcatgcat acccctcggt gttattgctg ctctgtgcac gcacgctcat tgtatcactt 1560 catcccagtg catgcactca cactggagcg attgctgctc ggtgcacgca cactcattgt 1620 atcacgtcag ctcagtggct gcacgcacac cggtgttatt gctgctcggt gcgtgcatgc 1680 acatcagtgt cgctgcagct cagtgcatgc acgctcattg cccatcgcta tccctgcctc 1740 tcctgctggc gctccccggg aggtgacttc aaggggaccg caggaccacc tcgggggtgg 1800 ggggagggct gcacacgcgg accccgctcc ccctccccaa caaagcactg tggaatcaaa 1860 aaggggggag gggggatgga ggggcgcgtc acacccccgc cccacaccct cacctcgagg 1920 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt 1980 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg 2040 ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga ggcggagagg tgcggcggca 2100 gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg 2160 ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgttgccttc gccccgtgcc 2220 ccgctccgcg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag 2280 gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg 2340 ctcgtttctt ttctgtggct gcgtgaaagc cttaaagggc tccgggaggg ccctttgtgc 2400 gggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg 2460 cccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc 2520 gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg gctgcgaggg 2580 gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcggc 2640 ggtcgggctg taaccccccc ctgcaccccc ctccccgagt tgctgagcac ggcccggctt 2700 cgggtgcggg gctccgtgcg gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc 2760 ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc cggggagggc tcgggggagg 2820 ggcgcggcgg ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct 2880 tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct ggcggagccg 2940 aaatctggga ggcgccgccg caccccctct agcgggcgcg ggcgaagcgg tgcggcgccg 3000 gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc 3060 atctccagcc tcggggctgc cgcaggggga cggctgcctt cgggggggac ggggcagggc 3120 ggggttcggc ttctggcgtg tgaccggcgg ggtttatatc ttcccttctc tgttcctccg 3180 cagcccccaa gcttaaggtg cacggcccac gtggggacta gtgccacc atg gcg ctc 3237 Met Ala Leu 1 ctg ctg tgc ttc gtg ctc ctg tgc gga gtc gcg gat ctc acc aga agt 3285 Leu Leu Cys Phe Val Leu Leu Cys Gly Val Ala Asp Leu Thr Arg Ser 5 10 15 ttg agt atc act act cct gaa cag atg att gaa aag gcc aaa ggg gaa 3333 Leu Ser Ile Thr Thr Pro Glu Gln Met Ile Glu Lys Ala Lys Gly Glu 20 25 30 35 act gcc tat ttg cca tgc aga ttt acc ctg ggt cca gaa gac cag ggg 3381 Thr Ala Tyr Leu Pro Cys Arg Phe Thr Leu Gly Pro Glu Asp Gln Gly 40 45 50 ccg ctg gac atc gag tgg ctg ctg tca cca gct gat aat cag aag gtg 3429 Pro Leu Asp Ile Glu Trp Leu Leu Ser Pro Ala Asp Asn Gln Lys Val 55 60 65 gat caa gtg att att tta tat tct gga gac aaa att tat gac gac tac 3477 Asp Gln Val Ile Ile Leu Tyr Ser Gly Asp Lys Ile Tyr Asp Asp Tyr 70 75 80 tac caa gat ctg aaa gga cga gta cat ttt aca agt aat gat ctc aaa 3525 Tyr Gln Asp Leu Lys Gly Arg Val His Phe Thr Ser Asn Asp Leu Lys 85 90 95 tca ggt gat gca tca ata aat gta aca aat cta cag ttg tca gat att 3573 Ser Gly Asp Ala Ser Ile Asn Val Thr Asn Leu Gln Leu Ser Asp Ile 100 105 110 115 ggc aca tat cag tgc aaa gtg aaa aag gct cct ggt gtt gga aat aag 3621 Gly Thr Tyr Gln Cys Lys Val Lys Lys Ala Pro Gly Val Gly Asn Lys 120 125 130 aag att cag ctg aca gtt ctt ctt aag cct tca ggt aca aga tgt tat 3669 Lys Ile Gln Leu Thr Val Leu Leu Lys Pro Ser Gly Thr Arg Cys Tyr 135 140 145 gtt gat gga tca gaa gaa att gga aat gac ttt aaa cta aaa tgt gaa 3717 Val Asp Gly Ser Glu Glu Ile Gly Asn Asp Phe Lys Leu Lys Cys Glu 150 155 160 cca aaa gaa ggt tca ctc cca tta cta tat gaa tgg cag aaa ttg tcc 3765 Pro Lys Glu Gly Ser Leu Pro Leu Leu Tyr Glu Trp Gln Lys Leu Ser 165 170 175 aat tca cag aag ctg ccc acc ttg tgg tta gca gaa atg act tca cct 3813 Asn Ser Gln Lys Leu Pro Thr Leu Trp Leu Ala Glu Met Thr Ser Pro 180 185 190 195 gtt ata tct gta aaa aat gcc tct act gaa tac tct ggg aca tac agc 3861 Val Ile Ser Val Lys Asn Ala Ser Thr Glu Tyr Ser Gly Thr Tyr Ser 200 205 210 tgt acc gtg aaa aac aga gtg ggc tct gat cag tgc ctg ctt cgc ctg 3909 Cys Thr Val Lys Asn Arg Val Gly Ser Asp Gln Cys Leu Leu Arg Leu 215 220 225 gat gtg gtt cct cct tca aat aga gct gga aca att gca gga gct gtt 3957 Asp Val Val Pro Pro Ser Asn Arg Ala Gly Thr Ile Ala Gly Ala Val 230 235 240 ata gga gtt ttg ctt gct cta gtg ctc att ggt ctt atc atc ttt tgc 4005 Ile Gly Val Leu Leu Ala Leu Val Leu Ile Gly Leu Ile Ile Phe Cys 245 250 255 tgt cgt taa tctagataag taatgatcat aatcagccat atcacatctg 4054 Cys Arg 260 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa 4114 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca 4174 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 4234 ccaaactcat caatgtatct tatcatgtct ggatccccgg gtaccgagct cg 4286 2 261 PRT porcine 2 Met Ala Leu Leu Leu Cys Phe Val Leu Leu Cys Gly Val Ala Asp Leu 1 5 10 15 Thr Arg Ser Leu Ser Ile Thr Thr Pro Glu Gln Met Ile Glu Lys Ala 20 25 30 Lys Gly Glu Thr Ala Tyr Leu Pro Cys Arg Phe Thr Leu Gly Pro Glu 35 40 45 Asp Gln Gly Pro Leu Asp Ile Glu Trp Leu Leu Ser Pro Ala Asp Asn 50 55 60 Gln Lys Val Asp Gln Val Ile Ile Leu Tyr Ser Gly Asp Lys Ile Tyr 65 70 75 80 Asp Asp Tyr Tyr Gln Asp Leu Lys Gly Arg Val His Phe Thr Ser Asn 85 90 95 Asp Leu Lys Ser Gly Asp Ala Ser Ile Asn Val Thr Asn Leu Gln Leu 100 105 110 Ser Asp Ile Gly Thr Tyr Gln Cys Lys Val Lys Lys Ala Pro Gly Val 115 120 125 Gly Asn Lys Lys Ile Gln Leu Thr Val Leu Leu Lys Pro Ser Gly Thr 130 135 140 Arg Cys Tyr Val Asp Gly Ser Glu Glu Ile Gly Asn Asp Phe Lys Leu 145 150 155 160 Lys Cys Glu Pro Lys Glu Gly Ser Leu Pro Leu Leu Tyr Glu Trp Gln 165 170 175 Lys Leu Ser Asn Ser Gln Lys Leu Pro Thr Leu Trp Leu Ala Glu Met 180 185 190 Thr Ser Pro Val Ile Ser Val Lys Asn Ala Ser Thr Glu Tyr Ser Gly 195 200 205 Thr Tyr Ser Cys Thr Val Lys Asn Arg Val Gly Ser Asp Gln Cys Leu 210 215 220 Leu Arg Leu Asp Val Val Pro Pro Ser Asn Arg Ala Gly Thr Ile Ala 225 230 235 240 Gly Ala Val Ile Gly Val Leu Leu Ala Leu Val Leu Ile Gly Leu Ile 245 250 255 Ile Phe Cys Cys Arg 260 3 1098 DNA porcine CDS (1)..(1098) 3 atg gcg ctc ctg ctg tgc ttc gtg ctc ctg tgc gga gtc gcg gat ctc 48 Met Ala Leu Leu Leu Cys Phe Val Leu Leu Cys Gly Val Ala Asp Leu 1 5 10 15 acc aga agt ttg agt atc act act cct gaa cag atg att gaa aag gcc 96 Thr Arg Ser Leu Ser Ile Thr Thr Pro Glu Gln Met Ile Glu Lys Ala 20 25 30 aaa ggg gaa act gcc tat ttg cca tgc aga ttt acc ctg ggt cca gaa 144 Lys Gly Glu Thr Ala Tyr Leu Pro Cys Arg Phe Thr Leu Gly Pro Glu 35 40 45 gac cag ggg ccg ctg gac atc gag tgg ctg ctg tca cca gct gat aat 192 Asp Gln Gly Pro Leu Asp Ile Glu Trp Leu Leu Ser Pro Ala Asp Asn 50 55 60 cag aag gtg gat caa gtg att att tta tat tct gga gac aaa att tat 240 Gln Lys Val Asp Gln Val Ile Ile Leu Tyr Ser Gly Asp Lys Ile Tyr 65 70 75 80 gac gac tac tac caa gat ctg aaa gga cga gta cat ttt aca agt aat 288 Asp Asp Tyr Tyr Gln Asp Leu Lys Gly Arg Val His Phe Thr Ser Asn 85 90 95 gat ctc aaa tca ggt gat gca tca ata aat gta aca aat cta cag ttg 336 Asp Leu Lys Ser Gly Asp Ala Ser Ile Asn Val Thr Asn Leu Gln Leu 100 105 110 tca gat att ggc aca tat cag tgc aaa gtg aaa aag gct cct ggt gtt 384 Ser Asp Ile Gly Thr Tyr Gln Cys Lys Val Lys Lys Ala Pro Gly Val 115 120 125 gga aat aag aag att cag ctg aca gtt ctt ctt aag cct tca ggt aca 432 Gly Asn Lys Lys Ile Gln Leu Thr Val Leu Leu Lys Pro Ser Gly Thr 130 135 140 aga tgt tat gtt gat gga tca gaa gaa att gga aat gac ttt aaa cta 480 Arg Cys Tyr Val Asp Gly Ser Glu Glu Ile Gly Asn Asp Phe Lys Leu 145 150 155 160 aaa tgt gaa cca aaa gaa ggt tca ctc cca tta cta tat gaa tgg cag 528 Lys Cys Glu Pro Lys Glu Gly Ser Leu Pro Leu Leu Tyr Glu Trp Gln 165 170 175 aaa ttg tcc aat tca cag aag ctg ccc acc ttg tgg tta gca gaa atg 576 Lys Leu Ser Asn Ser Gln Lys Leu Pro Thr Leu Trp Leu Ala Glu Met 180 185 190 act tca cct gtt ata tct gta aaa aat gcc tct act gaa tac tct ggg 624 Thr Ser Pro Val Ile Ser Val Lys Asn Ala Ser Thr Glu Tyr Ser Gly 195 200 205 aca tac agc tgt acc gtg aaa aac aga gtg ggc tct gat cag tgc ctg 672 Thr Tyr Ser Cys Thr Val Lys Asn Arg Val Gly Ser Asp Gln Cys Leu 210 215 220 ctt cgc ctg gat gtg gtt cct cct tca aat aga gct gga aca att gca 720 Leu Arg Leu Asp Val Val Pro Pro Ser Asn Arg Ala Gly Thr Ile Ala 225 230 235 240 gga gct gtt ata gga gtt ttg ctt gct cta gtg ctc att ggt ctt att 768 Gly Ala Val Ile Gly Val Leu Leu Ala Leu Val Leu Ile Gly Leu Ile 245 250 255 gtg ttt tgc tgt cat aaa aag cgc aga gaa gaa aaa tac gaa aaa gaa 816 Val Phe Cys Cys His Lys Lys Arg Arg Glu Glu Lys Tyr Glu Lys Glu 260 265 270 gtg cat cat gat atc agg gaa gac gtg cct cct ccg aag agc aga acg 864 Val His His Asp Ile Arg Glu Asp Val Pro Pro Pro Lys Ser Arg Thr 275 280 285 tcc act gcc aga agc tac ctc ggc agc aac cac tcg tcc ctg gga tcc 912 Ser Thr Ala Arg Ser Tyr Leu Gly Ser Asn His Ser Ser Leu Gly Ser 290 295 300 atg tct cct tcc aac atg gaa ggc tat tcc aag act cag tat aac cag 960 Met Ser Pro Ser Asn Met Glu Gly Tyr Ser Lys Thr Gln Tyr Asn Gln 305 310 315 320 gta cca agc gaa gac ttt gaa cgc gct cct cag agt cca act ctc ccg 1008 Val Pro Ser Glu Asp Phe Glu Arg Ala Pro Gln Ser Pro Thr Leu Pro 325 330 335 ctc gct aag gta gct gcc cct aat ctc agc cgg atg gga gcg gtg cct 1056 Leu Ala Lys Val Ala Ala Pro Asn Leu Ser Arg Met Gly Ala Val Pro 340 345 350 gtg atg att cca gcc cag agc aag gac ggg tcc ata gta taa 1098 Val Met Ile Pro Ala Gln Ser Lys Asp Gly Ser Ile Val 355 360 365 4 365 PRT porcine 4 Met Ala Leu Leu Leu Cys Phe Val Leu Leu Cys Gly Val Ala Asp Leu 1 5 10 15 Thr Arg Ser Leu Ser Ile Thr Thr Pro Glu Gln Met Ile Glu Lys Ala 20 25 30 Lys Gly Glu Thr Ala Tyr Leu Pro Cys Arg Phe Thr Leu Gly Pro Glu 35 40 45 Asp Gln Gly Pro Leu Asp Ile Glu Trp Leu Leu Ser Pro Ala Asp Asn 50 55 60 Gln Lys Val Asp Gln Val Ile Ile Leu Tyr Ser Gly Asp Lys Ile Tyr 65 70 75 80 Asp Asp Tyr Tyr Gln Asp Leu Lys Gly Arg Val His Phe Thr Ser Asn 85 90 95 Asp Leu Lys Ser Gly Asp Ala Ser Ile Asn Val Thr Asn Leu Gln Leu 100 105 110 Ser Asp Ile Gly Thr Tyr Gln Cys Lys Val Lys Lys Ala Pro Gly Val 115 120 125 Gly Asn Lys Lys Ile Gln Leu Thr Val Leu Leu Lys Pro Ser Gly Thr 130 135 140 Arg Cys Tyr Val Asp Gly Ser Glu Glu Ile Gly Asn Asp Phe Lys Leu 145 150 155 160 Lys Cys Glu Pro Lys Glu Gly Ser Leu Pro Leu Leu Tyr Glu Trp Gln 165 170 175 Lys Leu Ser Asn Ser Gln Lys Leu Pro Thr Leu Trp Leu Ala Glu Met 180 185 190 Thr Ser Pro Val Ile Ser Val Lys Asn Ala Ser Thr Glu Tyr Ser Gly 195 200 205 Thr Tyr Ser Cys Thr Val Lys Asn Arg Val Gly Ser Asp Gln Cys Leu 210 215 220 Leu Arg Leu Asp Val Val Pro Pro Ser Asn Arg Ala Gly Thr Ile Ala 225 230 235 240 Gly Ala Val Ile Gly Val Leu Leu Ala Leu Val Leu Ile Gly Leu Ile 245 250 255 Val Phe Cys Cys His Lys Lys Arg Arg Glu Glu Lys Tyr Glu Lys Glu 260 265 270 Val His His Asp Ile Arg Glu Asp Val Pro Pro Pro Lys Ser Arg Thr 275 280 285 Ser Thr Ala Arg Ser Tyr Leu Gly Ser Asn His Ser Ser Leu Gly Ser 290 295 300 Met Ser Pro Ser Asn Met Glu Gly Tyr Ser Lys Thr Gln Tyr Asn Gln 305 310 315 320 Val Pro Ser Glu Asp Phe Glu Arg Ala Pro Gln Ser Pro Thr Leu Pro 325 330 335 Leu Ala Lys Val Ala Ala Pro Asn Leu Ser Arg Met Gly Ala Val Pro 340 345 350 Val Met Ile Pro Ala Gln Ser Lys Asp Gly Ser Ile Val 355 360 365 

What is claimed is:
 1. A C-terminally truncated porcine CAR or a fragment or variant thereof which mediates adenoviral transduction.
 2. A C-terminally truncated porcine CAR according to claim 1 which is disclosed in SEQ ID NO: 2 or a fragment or variant thereof which mediates adenoviral transduction.
 3. A DNA sequence which encodes a C-terminally truncated porcine CAR according to claim
 1. 4. A DNA sequence which encodes a C-terminally truncated porcine CAR according to claim
 2. 5. A plasmid or vector construct that comprises a DNA molecule which expresses a porcine CAR or a fragment or variant thereof which mediates adenoviral transduction.
 6. A plasmid or vector construct that comprises a DNA which expresses a C-terminally truncated porcine CAR according to claim
 1. 7. A plasmid or vector construct that comprises a DNA which expresses a C-terminally truncated porcine CAR according to claim
 2. 8. Host cells into which a vector according to claim 5 has been introduced.
 9. Host cells into which a vector according to claim 6 has been introduced.
 10. Host cells into which a vector according to claim 7 has bene introduced.
 11. A method for generating transgenic rodents expressing or overexpressing porcine CAR comprising the steps of: (a) introducing a vector construct of claim 5 into an embryonic stem cell line; (b) selecting cells in which the introduced porcine gene has integrated; and (c) producing chimaeras which as chimaeric embryo may be implanted into a suitable pseudopregnant female foster animal and brought to term.
 12. A method of generating transgenic pigs overexpressing porcine CAR comprising the steps of: (a) introducing a vector construct of claim 5 into a non-quiescent differentiated pig cell or differentiated pig cell nucleus; (b) inserting said non-quiescent differentiated pig cell or non-quiescent differentiated pig cell nucleus into an enucleated pig oocyte, under conditions suitable for the formation of a nuclear transfer (NT) unit; (c) activating the resultant NT unit; (d) transferring said activated NT unit to a host pig such that the NT unit develops into a fetus; and optionally (e) developing the fetus to an offspring.
 13. A transgenic rodent obtained using the method according to claim
 11. 14. A transgenic pig obtained using the method according to claim
 12. 15. A method to test adenoviral transduction of an adenoviral gene delivery vector in a rodent animal model comprising the steps of: (a) removing organs, tissues or cells of transgenic rodents according to claim 13; (b) transducing in vitro said organs, tissues or cells with said adenoviral gene delivery vector; (b) transplanting said transduced organs, tissues or cells into the rodent animal model; and (d) assessing expression of the gene in said rodent animal model.
 16. A method for generating transgenic rodents expressing or overexpressing porcine CAR comprising the steps of: (a) introducing a vector construct of claim 6 into an embryonic stem cell line; (b) selecting cells in which the introduced porcine gene has integrated; and (c) producing chimaeras which as chimaeric embryo may be implanted into a suitable pseudopregnant female foster animal and brought to term.
 17. A method for generating transgenic rodents expressing or overexpressing porcine CAR comprising the steps of: (a) introducing a vector construct of claim 7 into an embryonic stem cell line; (b) selecting cells in which the introduced porcine gene has integrated; and (c) producing chimaeras which as chimaeric embryo may be implanted into a suitable pseudopregnant female foster animal and brought to term.
 18. A method of generating transgenic pigs overexpressing porcine CAR comprising the steps of: (a) introducing a vector construct of claim 6 into a non-quiescent differentiated pig cell or differentiated pig cell nucleus; (b) inserting said non-quiescent differentiated pig cell or non-quiescent differentiated pig cell nucleus into an enucleated pig oocyte, under conditions suitable for the formation of a nuclear transfer (NT) unit; (c) activating the resultant NT unit; (d) transferring said activated NT unit to a host pig such that the NT unit develops into a fetus; and optionally (e) developing the fetus to an offspring.
 19. A method of generating transgenic pigs overexpressing porcine CAR comprising the steps of: (a) introducing a vector construct of claim 7 into a non-quiescent differentiated pig cell or differentiated pig cell nucleus; (b) inserting said non-quiescent differentiated pig cell or non-quiescent differentiated pig cell nucleus into an enucleated pig oocyte, under conditions suitable for the formation of a nuclear transfer (NT) unit; (c) activating the resultant NT unit; (d) transferring said activated NT unit to a host pig such that the NT unit develops into a fetus; and optionally (e) developing the fetus to an offspring.
 20. A transgenic rodent obtained using the method according to claim
 16. 21. A transgenic rodent obtained using the method according to claim
 17. 22. A transgenic pig obtained using the method according to claim
 18. 23. A transgenic pig obtained using the method according to claim
 19. 24. A method to test adenoviral transduction of an adenoviral gene delivery vector in a rodent animal model comprising the steps of: (a) removing organ, tissues or cells of transgenic rodents according to claim 20; (b) transducing in vitro said organs, tissues or cells with said adenoviral gene delivery vector; (c) transplanting said trransduced organs, tissues or cells into the rodent animal model; and (d) assessing expression of the gene in said rodent animal model.
 25. A method to test adenoviral transduction of an adenoviral gene delivery vector in a rodent animal model comprising the steps of: (a) removing organ, tissues or cells of transgenic rodents according to claim 21; (b) transducing in vitro said organs, tissues or cells with said adenoviral gene delivery vector; (c) transplanting said trransduced organs, tissues or cells into the rodent animal model; and (d) assessing expression of the gene in said rodent animal model. 